Determination of the hydrophobic pulmonary surfactant protein SP-C

ABSTRACT

Methods for determining the strongly hydrophobic pulmonary surfactant protein SP-C, SP-C specific antibody and a reagent kit for carrying out the methods are described.

TECHNICAL FIELD

The invention relates to methods for determining the pulmonarysurfactant protein SP-C, to SP-C-specific antibodies and to theprovision of a reagent kit for carrying out the methods.

PRIOR ART

The lungs of all vertebrates contain a substance mixture called“pulmonary surfactant”. It has surface-active properties and reducessurface tension in the alveolar region of the lungs. In addition tophos-pholipids such as dipalmitoylphosphatidylcholine (DPPC) andphosphatidylglycerol (PG), the pulmonary surfactant contains proteins asfurther essential components. To date, four different surfactantproteins have been described, which are designated SP-A, SP-B, SP-C andSP-D, corresponding to the order in which they were discovered(Possmayer, F., A Proposed Nomenclature for PulmonarySurfactant-associated Proteins. Am. Rev. Respir. Dis. 1988, 138,990-998). The term SP stands for surfactant protein(surfactant-associated protein, SP).

SP-B and in particular SP-C are strongly hydrophobic proteins. The totalproportion of hydrophobic proteins in the pulmonary surfactant isapproximately 1% (Curstedt, T., Jörnvall, H., Robertson, B., Bergmann,T., Bergren, P.: Two Hydrophobic Low-Molecular-Mass Protein Fractions ofPulmonary Surfactant. Characterization and Biophysical Activity. Eur. J.Biochem. 1987, 168, 255-262).

The determination (detection and in particular quantification) of thesehydrophobic proteins in samples of pulmonary surfactant (for examplefrom lung lavage) frequently gives unsatisfactory results when thetechniques employed for hydrophilic proteins are used, if it is possibleat all. The methods which are customarily used for separating anddetermining hydrophilic proteins, such as, for example, “Westernblotting” or ELISA (Enzyme-Linked Immunosorbent Assay) techniques can beapplied to hydrophobic proteins only under certain conditions, since onthe one hand the “Western blot” per se is semiquantitative and, on theother hand, the use of an ELISA is very problematic, since it cangenerally only be carried out with proteins which are soluble in aqueoussystems. In most cases, only traces of the analytes to be quantified arepresent. Furthermore, in the samples to be analyzed, SP-B and SP-C arefrequently associated with other hydrophobic substances (for examplelipids), which renders quantification by customary methods even moredifficult. In the ELISA, the substance to be examined is usually notseparated prior to the determination from the other components which arecontained in the mixture. Van Eijk and co-workers (Van Eijk, M., DeHaas, C. G. M. and Haagsman, H. P.: Quantitative Analysis of PulmonarySurfactant Proteins B and C. Analytical Biochemistry 1995, 232, 231-237)describe a process for quantifying SP-C and SP-B in samples of pulmonarysurfactant. Extracts containing surfactant proteins are separated byhigh-pressure liquid chromatography, and SP-B or SP-C are detected andquantified by absorption at 228 nm. (The detection limits are at 1 μg ofSP-B and 4 μg of SP-C.)

For SP-B, a quantitative detection by ELISA technique is described(Krämer, H. J., Schmidt, R., Günther, A., Becher, G., Suzuki, Y.,Seeger, W.: ELISA Technique for Quantification of Surfactant Protein B(SP-B) in Bronchoalveolar Lavage Fluid. Am. J. Respir. Crit. Care Med.1995, 152, 1540-1544). An immunoassay for the determination of SP-C hashitherto not been described, apparently because the preparation ofSP-C-specific antibodies has not been successful (Beers, M. F., Wali,A., Eckenhoff, M. F., Feinstein, S. I., Fisher, J. H. and Fisher, A. B.:Am. J. Respir. Cell Mol. Biol. 1992, 7, 368-378).

DESCRIPTION OF THE INVENTION

It is an object of the invention to provide processes permitting thedetermination of pulmonary surfactant protein SP-C in a sample in asimple manner and with high sensitivity.

Surprisingly, we have succeeded in providing antibodies which arespecific for SP-C, and thus allow SP-C to be determined by animmunological method.

Accordingly, the invention provides processes for determining SP-C in asample, where the determination is carried out by an immunologicalmethod.

Further subject matters follow from the subclaims.

In the context of the invention, the determination of SP-C is to beunderstood as the detection and, in particular, the quantification ofSP-C.

In the context of the invention, the term “SP-C” is to be understood, inanalogy to the nomenclature proposed by Possmayer (Possmayer, F.: AProposed Nomenclature for Pulmonary Surfactant-associated Proteins. Am.Rev. Respir. Dis. 1988, 138, 990-998), as the “family” of surfactantproteins which is present in natural pulmonary surfactant or theamnionic fluid of mammals designated SP-C.

Furthermore, the term “SP-C” also includes chemically synthesized orrecombinantly prepared SP-C and modifications of SP-C, for example thosemodifications where one or more amino acids are missing or have beenreplaced by other amino acids. Chemically synthesized or recombinantlyprepared SP-C and modifications of SP-C are described, for example, inWO91/18015, WO91/00871, WO89/04326, WO93/21225 and also in WO95/32992.

SP-C is preferably understood as meaning the surfactant protein SP-Cwhich is present in human pulmonary surfactant or in human amnionicfluid.

In the context of the present invention, “immunological methods” areunderstood as meaning analytical methods based on immunochemistry, inparticular on an antigen-antibody reaction. Examples of immunologicalmethods include immunoassays such as radioimmunoassay (RIA), enzymeimmunoassay (EIA, combined with solid-phase technique: ELISA) or elseimmunofluorescence assays. The immunoassay is carried out by exposingthe sample to be investigated to an SP-C-binding antibody and detectingand quantifying the amount of antibody which binds to SP-C. In theseassays, detection and quantification is carried out directly orindirectly in a known manner. Thus, detection and quantification of theantigen-antibody complexes is made possible by using suitable labelswhich may be carried by the antibody directed against SP-C and/or by asecondary antibody directed against the primary antibody. Depending onthe type of the abovementioned immunoassays, the labels are, forexample, radioactive labels, fluorescent dyes or else enzymes, such asphosphatase or peroxidase, which can be detected and quantified with theaid of a suitable substrate.

In one embodiment of the invention, the immunological method is carriedout with the aid of a suitable solid phase. Suitable solid phases whichmay be mentioned include the customary commercial microtiter plates madeof polystyrene or membranes (for example made of polyvinylidenedifluoride, PVDF) which are customarily used for the ELISA technique.Surprisingly, it has been found that even chromatography plates aresuitable for use as solid phase in the process according to theinvention. The implementation of the process according to the inventionusing chromatography plates is hereinbelow also referred to asimmuno-TLC.

To carry out the process according to the invention, the sample isapplied to the solid phase. The sample is preferably a solution of SP-Cin a suitable solvent, and the solvent is evaporated after the samplehas been applied to the solid phase. To prepare a solution of the sampleto be investigated in a suitable solvent, it is advantageous to useorganic solvents or solvent mixtures which have proved to be suitablefor solubilizing hydrophobic proteins. Examples which may be mentionedare short-chain alcohols, in particular methanol, ethanol, 2-propanol(isopropanol) or n-propanol. Furthermore, in connection with immuno-TLC,mixtures of non-polar and polar solvents were found to be useful,suitable non-polar solvents being, in particular, chloroform, methylenechloride and toluene, and suitable polar solvents being short-chainalcohols. Chloroform/methanol mixtures may be mentioned as beingparticularly preferred.

If desired, small amounts of water and/or acid or base may be added tothe abovementioned solvents or solvent mixtures to improve the solventproperties.

If the SP-C content of a bronchoalveolar lavage (BAL) sample or a sampleof amnionic fluid is to be determined with the aid of immuno-TLC, it isadvantageous to transfer the SP-C prior to application to the solidphase from the aqueous phase (BAL sample, amnionic fluid) to a suitableorganic solvent or solvent mixture. This is expediently carried out byextraction with suitable organic solvents or solvent mixtures, forexample by the extraction method of Bligh-Dyer (Can. J. Biochem.Physiol. 1959, 37, 911-917) and subsequent Folch washing (J. Biol. Chem.1957, 226, 497-509).

If the SP-C content of a bronchoalveolar lavage (BAL) sample or a sampleof amnionic fluid is to be determined with the aid of the ELISAtechnique, it is advantageous to dilute the sample with a suitablewater-miscible solvent or solvent mixture. Suitable water-misciblesolvents or solvent mixtures which may be mentioned are, in particular,short-chain alcohols, such as ethanol and isopropanol, or mixturesthereof.

Moreover, it has been found to be expedient for the method according tothe invention to separate or remove interfering components in the sampleto be investigated on the solid phase prior to the immunologicaldetection of SP-C in the sample. This is particularly advantageous incases where in the samples to be investigated other components, such asother surfactant proteins or else lipids, are present in addition toSP-C.

If chromatography plates are used as the solid phase in the methodaccording to the invention, the sample can be separated by thin-layerchromatography. To this end, the sample is, after application andevaporation of the solvent, subjected to thin-layer chromatography.Suitable chromatography plates are all plates whose coating is suitablefor separating hydrophobic mixtures in organic media. The HPTLC platessold by Merck Darmstadt under the trade name Diol, which have a modifiedsilica matrix, have been found to be particularly suitable in thiscontext. Suitable solvents for the thin-layer chromatography ofhydrophobic proteins are organic solvents and solvent mixtures.Particularly expedient is the use of mixtures of non-polar and polarsolvents, suitable non-polar solvents being, in particular, chloroform,methylene chloride and toluene, and suitable polar solvents beingshort-chain alcohols, in particular methanol, ethanol and isopropanol.If desired, it is possible to add small amounts of water and/or acid orbase. Preference is given to mixtures of chloroform and methanol towhich small amounts of water and ammonia are added. The application ofthe samples to the plates and the practice of the separation are carriedout in a customary manner, for example by means of customary commercialapparatuses. The sample to be applied is preferably a solution of SP-Cin an organic solvent. In preparation for the immunological detection,the chromatography plates are dried after the separation by thin-layerchromatography.

It has surprisingly been found that, if the solid phase used is apolystyrene microtiter plate customarily used in the ELISA technique,interfering components in the sample can be removed after theapplication of the samples by one or more selective washing steps. Tothis end, BAL samples to be investigated are expediently diluted priorto application with a water-soluble organic solvent (for example analcohol such as isopropanol) and adjusted to a pH below 7 (preferably pH3-4). If desired, variable adsorption of the SP-C on the surface of theplate caused by inhomogeneities of the biological samples can beminimized. This can be effected, for example, after the application andthe drying of the sample—and before a washing—by re-dissolution in asuitable solvent, such as, for example, trifluoroethanol, followed by adrying step. Interfering components can be removed, after application ofthe samples and evaporation of the solvent, by washing with suitablesolvents. Thus, interfering lipids, for example, can be removed by oneor more washings with methanol.

In preparation for the immunological detection, the solid phase, ifdesired, is pretreated in a suitable manner. Unspecific binding sites onthe solid phase, for example, can be saturated using a suitable blockingsolution, such as gelatin, or a protein solution. The solid phase issubsequently incubated with a solution of the SP-C-specific antibody. Ifthis antibody is not labeled, detection and quantification can becarried out with the aid of a labeled secondary antibody whichrecognizes the primary antibody. To this end, excess primary antibody isremoved by washing and the plate is then incubated with a labeledsecondary antibody. After removal of excess antibody by washing,detection and quantification of the antigen-antibody complexes iscarried out with the aid of the label.

Labels which are suitable for the detection and quantification step andwhich can be carried by the primary or secondary antibody are known tothe person skilled in the art. Examples which may be mentioned includeradioactive labels, fluorescent dyes and, preferably, enzymes such asphosphatase or peroxidase, which permit calorimetric or chemoluminescentdetection. Depending on the label used, the subsequent detection orquantification is carried out in a known manner.

When carrying out the method according to the invention with the aid ofthe ELISA technique, the antigen-antibody complexes are preferablydetected and quantified via an enzyme-catalyzed color reaction using aperoxidase-conjugated antibody and ABTS(2,2′-azino-di[3-ethylbenzothiazolinesulfonate]) as substrate. However,it is also possible to use other chromogenic, chemoluminescent orfluorogenic methods known to the person skilled in the art for thedetection.

When carrying out the method according to the invention with the aid ofimmuno-TLC, the antigen-antibody complexes are preferably detected andquantified via an enzyme-catalyzed chemoluminescence reaction usingLuminol® as substrate. However, it is also possible to use otherchromogenic, chemoluminescent or fluorogenic methods known to the personskilled in the art for the detection.

As already mentioned, we have surprisingly succeeded in providingantibodies which are specific for SP-C, in particular for the SP-C whichis present in human pulmonary surfactant or amnionic fluid. Theinvention therefore also provides an antibody which is specific forSP-C, in particular specific for SP-C which is present in humanpulmonary surfactant or amnionic fluid. The antibody is furthermoreadvantageously characterized by the fact that it does not show any crossreactivity with other surfactant proteins such as SP-A and SP-B.

The SP-C-specific antibodies are preferably polyclonal antibodies.

The SP-C-specific antibodies are prepared analogously to processes knownto the person skilled in the art (as described, for example, inAntibodies: A Laboratory Manual. Eds. E. Hariow and D. Lane. Cold SpringHarbor Laboratory 1987).

The preparation of polyconal antibodies directed against SP-C isexpediently carried out by known immunization methods. Here, it has beenfound to be advantageous to use recombinant SP-C (herein-below alsoreferred to as rSP-C) as antigen in the immunization process, and toemploy larger amounts of antigen than are usually used, and to increasethe number of antigen administrations. In the immunization process,between 0.5 mg and 2 mg (preferably 1 mg) of rSP-C are expedientlyemployed per administration of antigen, where the number of antigenadministrations is advantageously between 5 and 7, preferably at aninterval of from three to six weeks (preferably four weeks).Furthermore, it has been found to be advantageous to use the rSP-C inthe form of insoluble particles (aggregates or precipitates), or coupledto a carrier molecule (for example protein or beads). Aggregates andprecipitates can be obtained in a customary manner, expediently asdescribed in the “Examples” section.

Suitable rSP-C for use in the immunization process which may bementioned includes recombinant human-identical dipalmitoylated SP-C(rhSP-C2Pam) or recombinant modifications of SP-C. Recombinantmodifications of SP-C which may be mentioned include, for example,non-palmitoylated human-identical SP-C (rhSP-C) or the SP-C modificationwith 34 amino acids described in the international patent applicationWO95/32992, where the amino acids in positions 4 and 5 of the amino acidsequence are phenylalanine and the amino acid in position 32 of theamino acid sequence is isoleucine (the stated positions within the aminoacid sequence are based on the amino acid sequence for the peptides ofthe formula I described in WO95132992). The preparation of this peptide,which is designated rSP-C34 (FF/1), by genetic engineering is likewisedescribed in WO95/32992.

The invention therefore also relates to a process for preparingpolyclonal antibodies directed against SP-C by immunization, inparticular against human SP-C, where the antigen component used for theimmunization is recombinant SP-C. By way of example, the preparation ofantibodies which are specific for SP-C, and an immunologicaldetermination of SP-C in a sample, are described below.

EXAMPLES 1. Preparation of Polyclonal Antibodies

1.1 Preparation of Antisera Against rSP-C

1 mg of rSP-C [rhSP-C2 Pam, rhSP-C or rSP-C34(FF/I)], dissolved inisopropanol/water (95:5), pH 4, was evaporated in a vacuum concentrator(Speedvac®) and resuspended in 0.5 ml of phosphate-buffered saline (PBS)by incubation with ultrasound (5 minutes) in an ultrasonic bath. Thisprocess gives aggregated rSP-C, which was used as antigen. Thesuspension was then mixed with 0.2 ml of Freund's adjuvant complete forthe basic immunization and with 0.5 ml of Freund's adjuvant incompletefor the booster injections. To achieve this, the solution was pushedback and forth 5-10× between two syringes linked by a canula. 0.2 mlportions of the emulsion were then injected subcutaneously into rabbits.The immunization scheme followed standard protocols: after the primaryimmunization, 5 booster injections were adminstered at intervals of 4weeks. Blood samples were taken in each case 10 days after the lastinjection to monitor the development of the titer. As soon as the titerwas satisfactory, 50 ml of blood were taken and serum was prepared bystandard methods.

It is also possible to use ABM-S (Antibody Multiplier Special) for thebasic immunization and AMB-N (Antibody Multiplier Normal) for thebooster injections, or to use other known adjuvants, for theimmunization. The use of Freund's adjuvant results, on the one hand, ina considerably higher antibody titer than when ABM adjuvant is used, onthe other hand, Freund's adjuvant induces an immune response in 100% ofthe animals, and when ABM is used, this is the case in only about 50% ofthe animals. Furthermore, the rSP-C can be coupled to a suitable carriermaterial, such as, for example, activated CH Sepharose, and then beadministered subcutaneously to the rabbits, with or without adjuvant,preferably with Freund's adjuvant.

All suitable animal species can be employed for preparing antibodies.Thus, in addition to rabbits, it is also possible to use chickens.

1.2 Determination of the Titer

In the individual sera, the titer was determined at different dilutionsusing Western blot analysis by standard methods. For gelelectrophoresis, the SDS/tricine method by Schägger and Jagow (Analyt.Biochem. 1987, 166:368-379) was used. Here, 0.1 μg of the appropriaterSP-C were separated in a 15% strength polyacrylamide gel, transferredto PVDF (polyvinylidene difluoride) membranes and incubated with thesera at dilutions of 1:1,000, 1:5,000, 1:10,000 and 1:25,000, 1:50,000,1:100,000 and 1:200,000. Bound primary antibodies were detected byperoxidase-coupled secondary antibodies (anti-rabbit IgG or anti-chickenIgY) via chemoluminescence using the ECL (Enhanced Chemiluminescence)system from Amersham (Brunswick). The titers varied from 1:10,000to >1:200,000. The detection limits in the Western blot varied from <10to 100 ng.

1.3 Specificity of the Antibodies Obtained: Cross Reactions With OtherPulmonary Surfactant Proteins

All antisera/antibodies reacted with the rSP-C employed in each case asantigen. Surprisingly, they cross reacted with natural dipalmitoylatedSP-C (hSP-C2 Pam; isolated from human or animal lung lavage), and alsowith other natural or modified rSP-Cs.

The antisera/antibodies do not react with other pulmonary surfactantproteins such as SP-A or SP-B. The antisera are therefore suitable fordetecting SP-C, in particular human SP-C, in solutions having an unknowncontent of SP-C, such as lung lavage or amnionic fluid, or for carryingout the detection on tissue sections using immunohistochemistry andimmunocytochemistry.

2. Immuno-TLC

2.1 Sample Preparation: Extraction of SP-C From Bronchoalveolar Lavages(BAL)

0.8 ml of BAL, 2.0 ml of methanol and 1.0 ml of chloroform are pipettedinto a centrifuge tube and thoroughly mixed using a vortex. Addition ofanother 1.0 ml of chloroform results in phase separation. After renewedmixing, 1.0 ml of ultrapure water are added, and the samples are oncemore mixed with the vortex for approximately 30 seconds. The samples arecentrifuged at 2400 rpm for 5 minutes. The bottom phases are (ifnecessary in two steps) transferred into Eppendorf caps and evaporatedto dryness using the Speed-Vac combination. In the meantime, the topphases are washed with 2.0 ml of chloroform/methanol/water (86/14/1 byvolume). The second bottom phases are in each case transferred into thesame Eppendorf caps of the first phase, centrifuged as above and alsoevaporated to dryness. The top phases are discarded. The extracts aretaken up in chloroform/methanol 70:30 (v/v).

2.2 Thin-layer Chromatography (TLC)

For thin-layer chromatography, HPTLC plates having a modified silicamatrix, as sold by Merck Darmstadt under the trade name Diol, were used.The samples were applied automatically onto the HPTLC plates using aLingomat IV (Camag, Berlin). After the samples had been applied, theplates were air-dried and chromatographed using a CHCl₃:MeOH mixture[CHCl₃/MeOH/25% strength NH₄OH/H₂O=32.5/15/1/2 (by volume)] as liquidphase. After the chromatography, the plates were dried.

2.3 Immunological Detection and Quantification

To saturate non-specific binding sites, the dried HPTLC plates wereincubated for 4 hours with 3% strength fish gelatin in PBS whichcontained 150 mM NaCl, 12 mM Na₂HPO₄ and 3 mM NaH₂PO₄ (ph 7.4). Theplates were then incubated overnight and, in the presence of the primarySP-C-specific antibody, shaken gently, usually at a dilution of1:10,000. Unbound antibodies were removed by repeated washing with TBS/Tfrom 4 mM tris-HCl, 100 mM NaCl, 0.05% Tween-20 (pH 7.4). Forhybridization with the primary antibody, the plates were incubated for 2hours with horseradish peroxidase-conjugated secondary antibody, at adilution of 1:100,000 in TBS/T. Unbound antibodies were removed asdescribed above by repeated washing of the plates with TBS/T.Immunoreactive complexes were visualized using ECL detection systems(consisting of Luminol® and enhancer) from Amersham Buchler. The plateswere exposed to an X-ray film (Hyperfilm Amersham) for 1-10 minutes.

2.4 Video-imaging of the X-ray Films and Evaluation by Computer

To quantify the immunocomplexes, the X-ray films were digitalized usinga Video-Imager (Cybertech, Berlin, Germany). The signal intensities onthe X-ray films were evaluated with the aid of a computer using thedensitometer software Diana (Raytest).

2.5 Quantification of SP-C

To determine pulmonary surfactant protein SP-C, BAL fractions wereextracted. In order to quantify the small amounts of SP-C, it isnecessary to separate the target protein from the large proportion oflipids. This is achieved by thin-layer chromatography. The SP-C in thefractions applied is quantified with the aid of standards.

3. ELISA Technique

3.1.1 Sample Preparation and Coating of the Microtiter Plates Using PBSBuffer

The carrier material used for the ELISA were microtiter plates with 96wells made of polystyrene (Polysorp® F96) with certificate, Nunc,Wiesbaden). The standard (human-identical rSP-C, c=300 μg/ml) wasdiluted with isopropanol/water (80:20, pH 3.0) in a series ofconcentrations from 40 ng/well to 312.5 pg/well (v=100 μl/well,corresponding to c=0.4-0.003125 μg/ml of rSP-C). The samples to bemeasured (bronchoalveolar lavages, BAL) were diluted withisopropanol/water (80.20, pH 3.0). Usually, 10 μl or 20 μl of BAL in atotal volume of 100 μl were pipetted into the wells. Each plate alsocarried an internal standard (BAL with a defined concentration of SP-C).All samples and standards were measured in duplicate. All dilutions werecarried out in 2 ml reaction vessels made of polypropylene (Eppendorf,Hamburg).

After the pipetting of the samples and standards, the microtiter plateswere incubated in a ventilated drying cabinet at 37° C. for 6 hours todryness. The variable adsorption of SP-C at the surface of the plates,which is caused by inhomogeneities of the biological samples, wasminimized by re-uptake in 100 μl of trifluoroethanol. After a furtherdrying step (37° C., 3 hours), the phospholipids, which were present inabout 50-100-fold excess relative to SP-C, were selectively removed bytwo successive washing steps. In the first step, 200 μl of methanol wereadded and the plate was incubated at room temperature with shaking (20minutes). The solvents were decanted off, and the plates were thenwashed once more with 200 μl of methanol, and the methanol wasimmediately decanted off. The plates were then washed three times with200 μl of PBS/0.5% Tween 20.

3.1.2 Sample Preparation and Coating of the Microtiter Plates UsingTris/Cl Buffer

All standard solutions and aqueous samples of SP-C were mixed with 80%2-propanol/20% water (v/v) pH 3.5. SP-C standards (human rSP-C, c=300μg/ml) were diluted to final concentrations of 0.4-0.003125 μg/ml.Aqueous samples (bronchoalveolar lavage fluids, BALF) were mixed 1:5(v/v) with 80% 2-propanol/20% water (v/v) pH 3.5. All dilutions wereperformed in 20 ml polypropylene vials (Eppendorf, Hamburg, Germany) inorder to avoid adsorption of SP-C to the vessel wall, before a volume of100 μl of each sample or standard was transferred to polystyrolemicrotiter plates (Polysorp® F96, with certificate, Nunc, Wiesbaden,Germany). BALF samples with known SP-C content were coprocessed on eachmicrotiter plate. All standards and samples were processed in duplicate.

Fluid removal was achieved by overnight evaporation at 37° C. Thesubsequent addition of 100 μl/well 1,1,1-trifluoroethanol, againfollowed by evaporation at 37° C. (3 h), optimized adsorption of thebiological samples to the plate. Phospholipids were selectively removedby two subsequent washing procedures with methanol. For this purpose,200 μl/well methanol was applied to the plates and incubated for 20 minat ambient temperature with gentle shaking. Following decantation of theorganic solvent, this step was repeated without further incubation.Immediately after decantation, wells were washed three times with 50 mMTris/Cl pH 7.6+0.5% Tween 20 (Sigma, Deisenhofen, Germany).

3.2.1 Immunological Detection and Quantification Using PBS Buffer

The ELISA system described below is based on the method developed byReinke et al. (Prostaglandins 1989, 37:577-586). To block excess bindingsites, the sample was incubated for 2 hours with PBS/1% bovine serumalbumin (BSA). After rinsing three times with PBS/0.5% Tween 20, theSP-C antiserum was applied. To this end, the antiserum was diluted1:2,000 in PBS/1% BSA, 200 μl of this solution were pipetted into eachwell, and the plate was incubated at room temperature for 12-15 hoursand subsequently once more washed three times (see above). 200 μl of a1:1000 dilution of biotinylated anti-rabbit antibody (donkey,Amersham-Buchler, Brunswick) in PBS/1% BSA were then applied, and theplate was incubated at room temperature for 2 hours. To remove excessantibody, the plate was washed three times. The sensitivity of the testwas then enhanced using the avidine/biotin peroxidase technique(AB-Komplex, Dako, Hamburg). To this end, a drop of avidine solution anda drop of biotinylated horseradish peroxidase solution were added to 5ml of PBS, and the mixture was equilibrated for 30 minutes. 200 μl of a1:50 dilution of this stock solution in PBSI1% BSA were incubated on theplates for 2 hours, and the plates were subsequently once more washedthree times (see above).

The enzymatic color development was initiated by addition of thesubstrate 2,2′-azino-di-[3-ethylbenzothiazolinesulfonate] (ABTS) [NGO,T. T.: Chromogenic substrates for enzyme immunoassay in nonisotopicimmunoassay. New York: Plenum Publishing Corporation, 1988, p. 57-84].To this end, 20 mg of ABTS and 10 μl of 30% H₂O₂ were dissolved in 30 mlof substrate buffer (60 mM sodium acetate trihydrate, 50 mM sodiumdihydrogen phosphate monohydrate pH 4.2) [PETERS, H. J.; BAUMGARTEN, H.;SCHULZE, M.: Monoklonale Antikörper-Herstellung und Charakterisierung.Berlin: Springer Verlag, 1985], and 200 μl of this solution werepipetted into the wells. After color development (2 hours, roomtemperature or overnight at 4° C.), the samples were evaluatedspectrophotometrically at 405 or 450 nm in an ELISA photometer (Reader400 V. 1.1, SLT, Crailsheim).

3.2.2 Immunological Detection and Quantification Using Tris/Cl Buffer

Nonspecific protein binding to wells was blocked with 200 μl 50 mMTris/Cl pH 7.6 buffer containing 1% (w/v) bovine serum albumin (BSA,Paesel and Lorei, Frankfurt/Main, Germany). After 2 h incubation atambient temperature, wells were washed three times with 50 mM Tris/Cl pH7.6+0.5% Tween 20 and a 1:2,000 dilution (v/v) of anti-SP-C antiserum in50 mM Tris/Cl pH 7.6+1% BSA was added. After 12-15 h incubation atambient temperature, wells were washed extensively as described aboveand incubated for 2 h with 200 μl of biotinylated anti-rabbit antibody(Amersham Buchler, Braunschweig, Germany; 1:1,000 in 50 mM Tris/Cl pH7.6+1% BSA). After removal of free antibody, incubation with 200 μl ofavidin-biotin-horseradish peroxidase complex (ABComplex, Dako, Glostrup,Denmark; stock solution according to the specification of the supplier,working solution 1:50 in 50 mM Tris/Cl pH 7.6+1% BSA) was performed foranother 2 h with subsequent washing. Enzymatic dye conversion wasinitiated by charging the wells with 200 μl2,2′-azino-di-[3-ethylbenzthiazolinsulfonate] (ABTS, Boehringer,Mannheim, Germany; 20 mg in 30 ml substrate buffer (60 mM sodiumacetate+50 mM NaH₂PO₄ pH 4.2)+10 μl 30% H₂O₂). After overnightincubation at 4° C., absorbance was measured at 450 nm.

3.3 Quantification of SP-C

The SP-C in the BAL samples was quantified by constructing a standardcurve by computer-supported cubic-spline interpolations using rSP-Cstandards which had been carried along.

Commercial Utility

The present method according to the invention allows the quantitativedetermination of the SP-C content of a sample in an advantageous manner.For example, it is possible to determine the SP-C content in humanpulmonary surfactant or amnionic fluid, or else to detect SP-C in tissuesections by immunohistochemistry and immunocytochemistry. The methodaccording to the invention can thus be employed advantageously fordiagnosing disease conditions associated with a pulmonary surfactantdeficit or a change in the composition of pulmonary surfactant (Güntheret al.: Surfactant Alterations in Severe Pneumonia, Acute RespiratoryDistress Syndrome and Cardiogenic Lung Edema. Am. J. Respir. Crit. CareMed. 1996, 153, 176-184; Seeger et al.: Alveolar Surfactant and AdultRespiratory Distress Syndrome. Clin. Investig. 1993, 71, 177-190; Cosmiet al.: Respiratory Distress Syndrome: Requirements of PerinatalDiagnosis, Prevention and Treatment. In Lachmann B. Ed.: SurfactantReplacement Therapy in Neonatal and Adult Respiratory Distress Syndrome,Springer Verlag 1988). Disease conditions which may be mentioned asexamples are Infant Respiratory Distress Syndrome (IRDS) or AdultRespiratory Distress Syndrome (ARDS).

The invention also provides a method for diagnosing IRDS or ARDS whichcomprises determining the SP-C content in human pulmonary surfactant orin amnionic fluid by an immunological method.

The invention also relates to a reagent kit for carrying out the methodaccording to the invention, wherein the reagent kit comprises anSP-C-specific antibody. Depending on how the method is carried out, thekit comprises other components. Other components which may be mentionedas examples are labeled secondary antibodies, buffer solutions, washingsolutions, reagents which are required for the detection step,chromatography plates or microtiter plates and organic solvents orsolvent mixtures.

What is claimed is:
 1. An isolated polyclonal antibody which is specificfor the human pulmonary surfactant protein SP-C.
 2. An isolatedpolyclonal antibody, obtainable by an immunization process in whichrecombinant human surfactant protein SP-C is used as antigen componentand the antibody is specific for SP-C which is present in humanpulmonary surfactant.
 3. A method for determining human pulmonarysurfactant protein SP-C in a sample by solid phase ELISA enzymeimmunoassay which comprises the steps of (i) providing a sample to theinvestigated, (ii) providing an antibody as claimed in claim 1, (iii)exposing the sample to be investigated to a solid phase and theantibody, and (iv) detecting and quantifying the amount of the antibodywhich binds to SP-C.
 4. A method for determining human pulmonarysurfactant protein SP-C in a sample, which comprises carrying out thedetermination by a solid phase ELISA enzyme immunoassay using anantibody as claimed in claim
 2. 5. The method as claimed in claim 3,wherein the solid phase is a chromatography plate and the sample isseparated on the chromatography plate prior to the enzyme immunoassaybeing carried out.
 6. The method as claimed in claim 4, wherein thesolid phase is a chromatography plate and the sample is separated on thechromatography plate prior to the enzyme immunoassay being carried out.7. The method as claimed in claim 4, wherein the solid phase is achromatography plate and the sample is separated on the chromatographyplate prior to the enzyme immunoassay being carried out.
 8. The methodas claimed in claim 3, wherein the solid phase is a microtiter plate. 9.The method as claimed in claim 4, wherein the solid phase is amicrotiter plate.
 10. A method for determining human pulmonarysurfactant protein SP-C in a sample, which comprises carrying out thedetermination by a solid phase enzyme immunoassay, wherein, in theenzyme immunoassay, the sample is exposed to a first, human pulmonarysurfactant protein SP-C-binding antibody, and the amount of boundantibody is measured using a second antibody carrying an enzyme label,where the measurement is carried out by an enzyme-catalyzed colourreaction or chemiluminescence.
 11. A reagent kit for carrying out amethod as claimed in claim 10, wherein the kit comprises a humanSP-C-specific antibody.